1. Field of the Invention
This invention relates to laboratory procedures for studying biological cells that have been physically deformed by mechanical stretching.
2. Description of the Prior Art
During the everyday physical activity of the human body, cells from tissues such as skeletal muscles, smooth muscles, and heart muscles are subjected to physical loads that cause the cells to stretch up to 10% or more. It is well established that mechanical stretch can have significant effects on cellular function, such as stimulation of intracellular Ca.sup.2+ release, increased gene expression, or release of paracrine growth factors. Published literature describing mechanical cell stretch and its effects on cellular function include papers by Perrone, C. E., et al., J. Biol. Chem. 270:2099-2106 (1995), Sadoshima, J., et al., Cell 75:977-984 (1993), and Wilson, E., et al., J. Clin. Invest. 96: 2364-2372 (1995). The effects of cell stretching are typically studied by growing the cells on a flexible membrane serving as a cell culture substrate, then deforming the membrane and, while maintaining the deformation, performing any of various procedures on the cells to study aspects of their biological function.
The literature reports the deformation of membranes in various ways for purposes of studying the effect on cell functions. Applying a uniaxial stretch is one example. Studies of cell and molecular responses to static and cyclic mechanical loading under uniaxial stretch are reported by Sadoshima, J., et al., cited above, Simpson, D. G., et al., Ann. NY Acad. Sci. 752:131-140 (1995), and Vandenburgh, H. H., Am. J. Physiol. 262 (Regulatory Integrative Comp. Physiol. 31):R350-R355 (1992). While the force in a uniaxial stretch is applied in a single direction, the resulting deformation of the substrate is actually a specific type of biaxial strain, since in addition to the stretching strain a compressive strain also occurs in directions transverse to the axis of the applied load, as reported by Hung, C. T., et al., J. Biomech. 27:227-232 (1994). Moreover, the strain distribution is nonhomogeneous, and significant shears are present near the clamped edges of the substrate. Thus, the result is a strain whose components and directions are not controlled and not fully quantified.
The literature also reports membrane deformation by biaxial stretch. Non-planar deformations, for example, have been used for achieving biaxial stretch. This is done by fixing the cells on a flexible circular substrate which is clamped to a holder, and applying a pressure differential across the substrate. The pressure differential inflates the substrate, causing it to bulge. As reported by Gilbert, J. A., et al., J. Biomech. 27:1169-1177 (1994), the strains produced in this type of device are nonhomogeneous in distribution, ranging in some cases between 0 and 30% over the surface of the substrate. Furthermore, the stretch differs from one axis to the next and shears may be present. These differences produce variations associated with the orientations of randomly plated polar cells, as well as variations in cellular responses. Also, focal plane differences in the substrate and the working distance requirements of an inverted microscope objective make it impractical to view cells that cultured on substrates that are inflated (caused to bulge) in this manner.
A further disadvantage of both uniaxial deformation and non-planar indentation is that the states of deformation that occur in these methods are not accurately defined. In addition, the substrate deformation is nonhomogeneous, and there is a lack of control over the type and magnitude of the strain imposed by the stretch. Quantifying cell deformation is essential for a complete understanding of the relationships between mechanical loads and cell function, in the same way that quantitative testing and theoretical analysis have been necessary for identifying relationships between structure and mechanical function in tissues and cells.
Biaxial stretch in a planar configuration can be achieved by pressing an object such as an indentation ring against the membrane surface. Devices operating in this manner are disclosed by Hung, C. T., et al., J. Biomech. 27:227-232 (1994) and by Schaffer, J. L., et al., J. Orthopaedic Res. 12:702-719 (1994). In these devices, the indentation ring and a stationary ring are pressed against opposite sides of the membrane, and in Hung et al., the indentation ring is the larger of the two. The circle of contact between the indentation ring and the membrane thus lies outside that of the stationary ring. The indentation ring is generally located below the membrane, and the stretch is controlled by pressing the indentation ring upward against the area of the membrane surrounding the stationary ring, consequently increasing the stretch across the stationary ring. The flat portion of the membrane that is stretched across the smaller stationary ring thus remains coplanar, but the larger movable indentation ring interferes with the access to the membrane by a microscope, particularly an inverted microscope. This precludes visualization of cells at high strains (i.e., strains of 1% or more).